Molecular mechanisms underlying bacterial resistance to ceftazidime/avibactam

Abstract Ceftazidime/avibactam (CAZ/AVI), a combination of ceftazidime and a novel β‐lactamase inhibitor (avibactam) that has been approved by the U.S. Food and Drug Administration, the European Union, and the National Regulatory Administration in China. CAZ/AVI is used mainly to treat complicated urinary tract infections and complicated intra‐abdominal infections in adults, as well as to treat patients infected with Carbapenem‐resistant Enterobacteriaceae (CRE) susceptible to CAZ/AVI. However, increased clinical application of CAZ/AVI has resulted in the development of resistant strains. Mechanisms of resistance in most of these strains have been attributed to bla KPC mutations, which lead to amino acid substitutions in β‐lactamase and changes in gene expression. Resistance to CAZ/AVI is also associated with reduced expression and loss of outer membrane proteins or overexpression of efflux pumps. In this review, the prevalence of CAZ/AVI‐resistance bacteria, resistance mechanisms, and selection of detection methods of CAZ/AVI are demonstrated, aiming to provide scientific evidence for the clinical prevention and treatment of CAZ/AVI resistant strains, and provide guidance for the development of new drugs. This article is categorized under: Infectious Diseases > Molecular and Cellular Physiology


| INTRODUCTION
Infections of patients with multidrug-resistant Gram-negative bacteria (MDRGNB) have become increasingly common. These bacteria have been associated with high morbidity and mortality rates, posing a serious threat to public health worldwide (Orsi et al., 2011). Carbapenems are considered one of the most powerful classes of therapeutic drugs for the 2.2 | Regional resistance surveillance of Enterobacteriaceae The 2012-2015 INFORM (the International Network for Optimal Resistance Monitoring, INFORM) resistance monitoring system showed that the overall resistance rate of Enterobacteriaceae to CAZ/AVI was low in different regions of the world, with resistance rates in Latin America and Europe is only 0.3%, while the resistance rate in the Asia Pacific is relatively higher, at 1.0% (Karlowsky et al., 2018(Karlowsky et al., , 2019K. M. Kazmierczak, de Jonge, et al., 2018a). By 2015-2017, the resistance rate of Enterobacteriaceae to CAZ/AVI in the Asia-Pacific region increased to 1.9%, and further investigation of the resistance rate of carbapenem nonsusceptible Enterobacteriaceae to CAZ/AVI in different regions showed that Oceania had the highest CAZ/AVI resistance rate of 88.9%, followed by Asia (51.7%), Africa-Middle East (49.2%), Europe (23.2%), and the lowest resistance rate was found in Latin America (12.5%; Spiliopoulou et al., 2020). By 2017-2019, carbapenem nonsusceptible Enterobacteriaceae bacteria in Latin America increased their resistance rate to CAZ/AVI nearly 1-fold to 25.3% (Karlowsky et al., 2021). In addition, global resistance surveillance by the ATLAS project from 2012 to 2016 showed that carbapenem-resistant Enterobacteriaceae had a resistance rate of 57.7% to CAZ/AVI, followed by CREC with a resistance rate of 27.7% to CAZ/AVI (H. Zhang, Xu, et al., 2020). The distribution of resistance rate of CRE to CAZ/AVI in different regions was as follows: the highest resistance rate to CAZ/AVI was 66.6% in the Asia-Pacific region, followed by Africa-Middle East (39.5%), Europe (28.0%), and Latin America (18.5%), whose trend was consistent with the 2015-2017 INFORM surveillance system showing the distribution of resistance rates of carbapenem nonsusceptible Enterobacteriaceae to CAZ/AVI (Table 1). These results suggest that CRE resistance to CAZ/AVI varies by region and that the differences in CAZ/AVI resistance rates are likely due to the different percentages of MBLs in different regions (Kiratisin et al., 2021;Spiliopoulou et al., 2020).
F I G U R E 2 Example of an irreversible reaction between a β-lactam and a nucleophilic serine enzyme. The figure shows the reaction of a penicillin with a penicillin binding protein (transpeptidase) to yield a stable acyl-enzyme complex, which subsequently undergoes slow hydrolysis.  2.3 | Regional resistance surveillance of P. aeruginosa Pseudomonas aeruginosa has a higher resistance rate to CAZ/AVI compared to Enterobacteriaceae. INFORM resistance surveillance system in 2012-2015 showed the highest resistance rate to CAZ/AVI in Latin America at 12.6%, followed by Europe (7.6%) and the Asia Pacific (7.4%; Karlowsky et al., 2018Karlowsky et al., , 2019K. M. Kazmierczak, de Jonge, et al., 2018b). In recent years, the resistance rate of P. aeruginosa to CAZ/AVI has been relatively stable across continents, with 13.1% in Latin America, 7.7% in Europe, and 7.3% in the Asia Pacific (Karlowsky et al., 2021;K. M. Kazmierczak, de Jonge, et al., 2018b;Ko & Stone, 2020;Lee et al., 2022). Among carbapenemase-producing P. aeruginosa, the resistance rate to CAZ/AVI was high in all continents: 92.3% in Africa-Middle East, 90.6% in the Asia Pacific and Europe, and 82.9% in Latin America (Kiratisin et al., 2021), showing that carbapenemase production correlates strongly with CAZ/AVI resistance (Table 1).  Yin et al., 2019). Taken together, these findings indicate that MBL-producing strains account for a high proportion of CAZ/AVI-resistant strains. By contrast, the main mechanisms underlying resistance to CAZ/AVI in Gram-negative bacteria that do not produce MBL include: (1) β-lactamase variants; (2) changes in bacterial membrane permeability; (3) increased expression of efflux pumps; and (4) mutation of PBPs .

| β-Lactamase variants
β-Lactamase variants are an important mechanism of bacterial resistance to CAZ/AVI. Alteration of key residues at the active sites of serine β-lactamases can increase their MIC values for CAZ/AVI significantly ( β-lactamases are dependent on several highly conserved sequences responsible for recognition of, and reactions with, antibiotics. These sequences include the Ω-loop, consisting of amino acids 164-179. Glu166 and Asn170 are involved in the acylation and deacylation of antibiotics by β-lactamases. The Ω-loop acts as a structural domain unit that interacts with antibiotics, forming the binding cavity for β-lactamase-substrate action. This allows the carbonyl on the β-lactam ring of the antibiotic to polarize, forming an acylated enzyme complex. The amino acid substitution in the KPC enzyme alters the structure of the binding cavity, thereby reducing resistance to the binding of CAZ and the active site of the enzyme, thereby increasing β-lactamase binding and hydrolysis of CAZ, an important mechanism of bacterial resistance to CAZ/AVI. Individual amino acid substitutions, particularly at positions 164, 167, 169, and 179 of the Ω-loop, increase the affinity of the β-lactamase to CAZ and increase its hydrolysis . In addition, amino acid substitutions in the Ω-loop can reduce the binding of β-lactamase to AVI, weakening the inhibitory effect of AVI on β-lactamase and increasing hydrolysis of CAZ (F. Compain et al., 2020aCompain et al., , 2020b. Alterations in β-lactamase expression can also lead to CAZ/AVI resistance (Humphries et al., 2015).

| Alterations in KPC enzymes
KPC-2 and KPC-3, which are distributed widely worldwide, are not only present in K. pneumoniae, but have also been detected in other Enterobacteriaceae, including Acinetobacter baumannii and P. aeruginosa (Bush & Bradford, 2019).
The main mechanisms of resistance associated with KPC are mutations in the bla KPC gene and increased bla KPC expression.
Experimentally, mutations have been introduced into bla KPC by site-directed mutagenesis, with the plasmids containing mutant bla KPC-3 used to transform E. coli DH 5α by heat shock. The MIC value of native bla KPC toward CAZ/AVI is 0.5/4 mg/L, but is altered by mutation to 16/4 mg/L for Asp179Tyr/Thr243Met, 8/4 mg/L for Asp179Tyr and Thr243Met, and 4/4 mg/L for the 165-166 Glu-Leu insertion (Haidar et al., 2017). The greater change in MIC value for the Asp179Tyr mutation than for the 165-166 Glu-Leu insertion or the Thr243Met mutation in CAZ/AVI suggests that the Ω-loop plays a central role in maintaining the stability of the KPC enzymes. The Ω-loop is formed by a salt bridge between Asp at position 179 and Arg at position 164. The less stable Asp179Tyr mutant has increased affinity for CAZ, and restricted binding to AVI. The 167-168Glu-Leu deletion in the Ω-loop of KPC-3 was also found to increase the MIC value of CAZ/AVI from 1/4 to 16/4 mg/L, resulting in a phenotypic shift from susceptible to resistant (Antinori et al., 2020).
Similarly, KPC-2 mutants containing Arg164Ala, Arg164Pro, Asp179Ala, Asp179Gln, and Asp179Asn amino acid substitutions are all resistant to CAZ/AVI, with MIC values of 16/4, 64/4, 64/4, 32/4, and 64/4 mg/L, respectively. To further study the mechanism by which KPC mutants increase MIC values for CAZ/AVI, the β-lactamase kinetics and the inhibitory activity of AVI were evaluated in the Arg164Ala and Asp179Asn mutants. Evaluation of their catalytic activities using nitrocefin kinetic assays showed that the Arg164Ala variant had a 10-fold higher K m for nitrofecin than wild-type KPC-2. Rapid-mixing stopped-flow kinetic assays of KPC-2 and the Asp179Asn variant on a 1.5 ms timescale revealed that the Asp179Asn variant revealed a phenomenon called "burst". This "burst" contributed to the resistance of the enzyme to CAZ, and its inability to bind to penicillin binding proteins (PBPs). Evaluation of the K i app and K 2 /K values of the Arg164Ala and Asp179Asn variants showed that similar concentrations of AVI were needed to fully inhibit both. These findings suggested that increased affinity for CAZ may prevent the binding of AVI to enzymes with Ω-loop substitutions, thereby increasing MIC values for CAZ/AVI rather than reducing AVI inhibition . The Asp179Tyr variant of KPC-2, called KPC-33, which is induced by selective pressure asserted by CAZ/AVI, showed an increase in the MIC value for CAZ/AVI from 0.125/4 to 16/4 mg/L, while at the same time restoring susceptibility to carbapenem antibiotics, which is useful for treatment of infection by CAZ/AVI-resistant strains (P. Zhang, Shi, et al., 2020). A strain containing the Ala172Thr variant of KPC-3, called KPC-39, was found to have a MIC value for CAZ/AVI of 12/4 mg/L. A study of its molecular structure showed that the Ala172Thr substitution enlarged the spatial structure of the Ω-loop active site, enabling it to better accommodate CAZ and increase the affinity between CAZ and the variant enzyme, resulting in a resistant phenotype (Jousset et al., 2021).
In addition to the Ω-loop amino acid substitutions, some other substitutions can alter the affinity of the enzyme for CAZ, leading to CAZ/AVI resistance. The Asp163Gly substitution adjacent to the Ω-loop in KPC-3 increased the MIC of K. pneumoniae clinical isolates for CAZ/AVI from 1/4 to 32/4 mg/L; the 181_182Ser-Ser insertion increased the MIC from 1/4 to 64/4 mg/L; the 180_181Ser insertion increased the MIC of Enterobacter cloacae for CAZ/AVI from 0.25/4 to 32/4 mg/L, whereas the 183_184Arg-Ala-Val-Thr-Thr-Ser-Ser-Pro insertion increased the MIC from 0.5/4 to 128/4 mg/L . A novel CAZ/AVI-resistant mutant, KPC-14, containing a two-amino acid deletion in KPC-2 (242_243Gly-Thr) and isolated from patients in New York in 2020, has a MIC value for CAZ/AVI of >16/4 mg/L. This enzyme was significantly more efficient than KPC-2 at catalyzing CAZ, but did not affect AVI inhibitory properties, suggesting that CAZ/AVI resistance may be due to an increase in β-lactamase hydrolysis of CAZ rather than a decrease in its inhibition by AVI (Niu et al., 2020). In addition, KPC-3 variants from clinical isolates of K. pneumoniae in Switzerland were resistant to CAZ/AVI Gregory et al., 2010;Mueller et al., 2019;Poirel et al., 2020). These included KPC-41, with a 269_270 Pro-Asn-Lys insertion and a MIC >128/4 mg/L; KPC-50, with a 276_277Glu-Ala-Val insertion and a MIC of 256/4 mg/L; KPC-8, with a Val240Gly substitution and a MIC of 32/4 mg/L; and KPC-23, with a Val240Ala substitution and a MIC of 16/4 mg/L. In addition, combinations of these alterations, such as KPC-64, a KPC-3 variant with a Ser181 insertion, and Tyr244Ala substitution had a MIC >256/4 mg/L and were resistant to CAZ/AVI (Venditti et al., 2021).
Bacterial resistance to CAZ/AVI is due not only to the altered affinity of KPC variants for CAZ, but also to the altered ability of AVI to inhibit these enzymes. A mutant containing a KPC-2 variant with a Asp179Tyr substitution (Barnes et al., 2017;Compain & Arthur, 2017;Hemarajata & Humphries, 2019) was found to have a CAZ/AVI MIC of 32/4 mg/L, whereas the CAZ/AVI MIC for wild-type KPC-2 was 1/4 mg/L. The Asp179Tyr substitution reduced the AVI rate constant for KPC-2 inhibition by approximately 70,000-fold, suggesting that the resistance of this variant to CAZ/AVI was also partly due to a significant reduction in the ability of AVI to inhibit the enzyme.
Culture of strains susceptible to CAZ/AVI in LB medium containing CAZ/AVI for 50 generations resulted in the generation of several variants of KPC-2, such as a 165_166 Gln-Leu insertion, a Leu169Pro insertion plus a Ser181 insertion, and a 166_167 Gln-Leu deletion. Introduction of these bla KPC mutations into a wild-type strain susceptible to CAZ/AVI increased the MIC values for CAZ/AVI from 2/4, 4/4, and 0.25/4 mg/L to 16/4, 128/4, and 32/4 mg/L, respectively, but did not affect MIC values for CAZ significantly, suggesting that these variants mainly affect the inhibitory activity of AVI .
On other hand, increased expression of bla KPC can also contribute to CAZ/AVI resistance. The first KPC-3-producing K. pneumoniae strain resistant to CAZ/AVI did not contain mutations in the bla KPC-3 gene (Humphries et al., 2015). Real-time quantitative PCR analysis, however, found that expression of the bla KPC-3 gene was 3.8 ± 0.2-fold higher in this resistant strain than in susceptible strains . Moreover, bla KPC-3 gene expression was found to be 2.5-fold or 1.5-fold higher in resistant strains than in wild-type strains (Gaibani et al., 2020). A study comparing 12 strains resistant to CAZ/AVI carrying wild-type bla KPC-2 with five strains susceptible to CAZ/AVI, also carrying bla KPC-2 , found that the number of copies of bla KPC-2 was 2.5-fold higher, and its expression 2.7-fold higher, in resistant than in susceptible strains (P. Zhang, Shi, et al., 2020). Evaluation of two CAZ/AVI-resistant strains isolated from a patient who had not been treated with CAZ/AVI showed that both strains carried two copies of the transposon Tn4401a encoding the KPC-3 enzyme, thereby increasing the number of bla KPC gene copies as well as its expression in the bacteria (Coppi et al., 2020). Moreover, increased expression of bla KPC resulted in the incorporation of structural changes in the outer membrane proteins, thereby reducing outer membrane protein pore size and permeability, leading (ultimately) to reduced resistance to CAZ/AVI (Coppi et al., 2020). In addition, the increase in bla KPC-3 expression was due to the transposition of the Tn4401 transposon carrying bla KPC-3 onto the plasmid plncX3, thereby increasing the number of bla KPC-3 copies . Taken together, these findings show that increases in the number of copies and expression of bla KPC are associated with CAZ/AVI resistance, and are most likely caused by an increase in the number of plasmids carrying bla KPC or an increase in the number of copies of bla KPC on the same plasmid.
3.1.2 | bla CTX-M and bla SHV mutations lead to CAZ/AVI resistance CTX-M enzymes and SHV enzymes are not common in strains resistant to CAZ/AVI; however, they deserve attention. For example, a K. pneumoniae strain carrying wild-type bla CTX-M-14 had a MIC value for CAZ/AVI of 1/4 mg/L, whereas a K. pneumoniae strain showing both high expression of bla OXA-48 and a mutant bla CTX-M-14 41704264 encoding a CTX-M-14 enzyme with Pro170Ser and Thr264Ile substitutions, showed an increase in the MIC value from 1/4 to 32/4 mg/L. To further confirm the role played by bla CTX-M-14 41704264 , its introduction into E. coli TOP 10 increased the MIC value for CAZ/AVI by 16-fold, from 0.5/4 to 8/4 mg/L, and increased the MIC value for CAZ by 64-fold, from 4/4 to 256/4 mg/L, suggesting that resistance of the mutant to CAZ/AVI is likely due to increased hydrolysis of CAZ. An Asp182Tyr substitution in the CTX-M-15 enzyme is also associated with decreased susceptibility to CAZ/AVI, with a 16-fold increase in its MIC value for CAZ/AVI (Both et al., 2017;Dulyayangkul et al., 2020;Livermore et al., 2018). In addition, a variant of CTX-M-15 harboring two amino acid substitutions, Gln169Leu and Gly130Ser, induced resistance to CAZ/AVI in an E. coli strain susceptible to this combination (Compain et al., 2018). The Km value of WT CTX-M-15 was 10-fold higher than that of the mutant enzyme, with the mechanism of resistance associated with increased affinity of the mutant enzyme for CAZ. CTX-M-15 includes several conserved sequences, such as 166_170 Glu-Pro-Thr-Leu-Asn on the 19-residue Ω-loop constituting the floor of active site. In addition, the conserved sequence 130_132 Ser-Asp-Asn is situated on a short loop in the allalpha domain, where it forms one side of the catalytic cavity (Compain et al., 2018). The structural features of CTX-M-15 suggest that the Pro170Ser substitution enhances the ability of the mutant enzyme to hydrolyze CAZ by altering the conformation of the Ω-loop, thereby enabling CAZ to more easily enter the active site of the mutant enzyme. In addition, the Gly130Ser substitution may alter the structure of the catalytic cavity, enabling CAZ to more easily enter the catalytic cavity, as well as increasing the affinity between the variant enzyme and CAZ, leading ultimately to CAZ/AVI resistance.

| ampC mutations cause CAZ/AVI resistance
Among the class C β-lactamases, AmpC enzyme variants are the most common. The wild-type AmpC enzyme has a MIC value for CAZ/AVI of 4/4 mg/L, whereas deletion of amino acid residues from its Ω-loop, such as the 245_249 Asp-Ala-Glu-Gly-Tyr and 238_244 Arg-Val-Gly-Pro-Gly-Pro-Leu deletions, increase the MIC values for CAZ/AVI from 4/4 mg/L to 256/4 and 64/4 mg/L, respectively (Lahiri et al., 2015).
The Asn346Tyr amino acid substitution in AmpC cloacae , DHA-1, and PDC-5 affects the ability of AVI to inhibit these enzymes. In the wild-type enzyme, the carboxamide at Asn346 forms hydrogen bonds with avibactam sulfonate. The amino acid substitution at this position results in loss of this interaction, which is likely reinforced by steric hindrance due to the bulky Tyr side chain, resulting in a significant reduction in carbamoylation activity (Compain et al., 2020a(Compain et al., , 2020b. In addition, the Gly183Asp amino acid substitution, caused by a nonsynonymous G548A nucleotide mutation in bla PDC-5 , results in resistance to CAZ/AVI, thereby increasing the MIC of P. aeruginosa against CAZ/AVI from 4/4 to ≥32/4 mg/L (MacVane et al., 2017). Moreover, AmpC enzyme variants containing Gly176Arg, Gly176Asp, Arg168Pro, Asn366Tyr, and Arg168His amino acid substitutions, generated in Enterobacter cloacae by stepwise induction, were resistant to CAZ/AVI, with MIC values ranging from 0.5/4 mg/L in wild-type to 64/4, 16/4, 16/4, and 32/4 mg/L, respectively, in the mutants. However, the mechanisms of resistance associated with these amino acid substitutions differed. Specifically, the Arg168Pro substitution reduced the affinity of the AmpC enzyme for AVI, and eliminated the synergy between CAZ and AVI, whereas the Arg168His and Gly176Arg/Asp substitutions increased the affinity of the enzyme for CAZ (Lahiri et al., 2014). Because the Asn346 residue is key to AVI binding, the Asn346Tyr substitution reduces the ability of AVI binding, resulting in resistance to CAZ/AVI (Lahiri et al., 2014). Moreover, CAZ/AVI resistance in ESBL-producing mutants is associated mostly with changes in the efflux pump, membrane permeability, or β-lactamase expression (Livermore et al., 2018). Compared with CYM-2, CYM-172 has a 290_292 Lys-Val-Ala deletion and a Asn346Ile substitution. Introduction of this mutant gene into E. coli DH5α through plasmid conjugation increases the MIC value for CAZ/AVI to 16/4 or 32/4 mg/L. CAZ/AVI resistance may be due to a change in CMY structure caused by the deletion of amino acids in the R2-loop of the enzyme, which may increase CAZ hydrolysis. Additionally, the Asn346Ile substitution results in steric hindrance between the Ile side chain and the sulfate group of AVI, thereby altering the binding affinity of the latter for the enzyme and resulting in CAZ/AVI resistance (M. Xu, Zhao, et al., 2021).
In addition to mutations and increased expression of the ampC gene, AVI induces AmpC enzyme activity. To test the ability of AVI to induce AmpC enzyme activity, strains of E. cloacae, Citrobacter freundii, and P. aeruginosa were incubated with 1, 4, and 32 mg/L AVI. At a concentration of 32 mg/L, AVI strongly induced expression of the AmpC enzyme in two of five E. cloacae and in two of five P. aeruginosa strains, with the enzyme content being thousands-fold higher than prior to induction. These findings suggest that the affinity of AmpC variants for AVI is reduced, but that these enzymes can be induced by AVI, resulting in CAZ/AVI resistance (D. M. Livermore, Jamrozy, et al., 2017). 3.1.4 | bla OXA mutations lead to CAZ/AVI resistance The MICs of 265 OXA-48-producing strains (collected by the International Optimal Resistance Monitoring Network between 2012 and 2015) for CAZ/AVI ranged from 0.03 to >128 mg/L, with 7.5% of these strains being resistant to CAZ/AVI (K. M. Kazmierczak, Bradford, et al., 2018). The presence of both Pro68Ala and Tyr211Ser amino acid substitutions in OXA-48-producing strains resulted in decreased susceptibility to CAZ/AVI. Enzyme kinetics showed that, compared with wild-type OXA-48, the variant enzyme had >20-fold higher ability to hydrolyze CAZ, and a >5-fold reduction in the inhibitory activity of AVI, suggesting that both are responsible for CAZ/AVI resistance. Molecular modeling showed that the hydrogen bond network mediated by water molecules was altered in the variant enzyme. In wild-type enzymes, Tyr211OH and Thr234O, and Pro235O and Met236O, form hydrogen bonds with central water molecules. In the variant enzyme, however, Ser211 cannot participate in the same H-bond network because of its lessspace-filling properties than Tyr211. The loss of H-bonds could enhance enzyme flexibility, which resulted in increased hydrolysis of CAZ. Moreover, the loss of aromatic stacking resulting from the Tyr211Ser amino acid substitution reduced AVI inhibitory activity (Fröhlich et al., 2019). In addition, OXA-539, a variant of OXA-2 with a Asp149 repeat, was found to be resistant to CAZ/AVI (Fraile-Ribot et al., 2017), as were several Acinetobacter baumannii strains carrying class D β-lactamases, such as OXA-23, OXA-40, OXA-58, and OXA-66. The inability of AVI to penetrate into their cell membranes prevents AVI from inhibiting these carbapenemases (Yoshizumi et al., 2015).

| CAZ/AVI resistance caused by other β-lactamase variants
In October 2019, an outbreak of CAZ/AVI-resistant KPC-2-producing K. pneumoniae was reported in Athens, Greece. Resistance of the strain was due to a variant of the Vietnamese extended-spectrum β-lactamase-1 (VEB-1). Compared with VEB-1, which had a MIC value for CAZ/AVI of 0.25/4 mg/L, this variant, VEB-25, with a Lys234Arg amino acid substitution, had a MIC value of 16/4 mg/L. Resistance to CAZ/AVI was due to interference by Arg of the ability of AVI to bind to the active site of the enzyme, thereby attenuating the inhibitory effect of AVI (Galani et al., 2020).

| CAZ/AVI resistance associated with changes in bacterial membrane permeability
The bacterial outer membrane proteins OmpF and OmpC, and their homologous proteins, play a role in CAZ/AVI resistance, although these proteins do not constitute the main pathway by which AVI passes through the cell walls of Enterobacteriaceae (Pagès et al., 2015).
A study investigating whether mutations in outer membrane proteins affect CAZ/AVI antibacterial activity found that the IS 5 insertion in ompK36, or the 134_135 Gly-Asp insertion in OmpK36, when combined with ESBL such as SHV, TEM, and CTX-M, are associated with decreased susceptibility of K. pneumoniae to CAZ/AVI (Shields et al., 2015). The decreased susceptibility of KPC-producing K. pneumoniae to CAZ/AVI was not only related to high expression of the bla KPC gene, but also to loss of OmpK35, as confirmed by SDS-PAGE. This indicates that the absence of OmpK35 contributes to the barrier of CAZ to permeate cells (Pagès et al., 2015). The genetic mechanism underlying OmpK35 deficiency varies by Sequence Type. For example, OmpK35 deficiency in ST11 strains may be due to early frameshift mutations and premature termination codons, whereas decreased OmpK35 expression in ST15 strains is associated with negative regulators of micF and ompR. MicF plays an important role in the transcriptional regulation of OmpK35 expression, whereas ompR negatively regulates the expression of OmpK35, either directly or through micF (Delihas & Forst, 2001).
The increased clinical use of CAZ/AVI has given rise to CAZ/AVI-resistant strains of K. pneumoniae. The first CAZ/AVI-resistant KPC-3-producing strain of K. pneumoniae, KP1244, was compared with the CAZ/AVI-susceptible strain KP1245. Whole-gene sequencing showed that both KP1244 and KP1245 have an Arg191Leu substitution in OmpK36, with KP1244 also having a Thr333Asn substitution in OmpK36. A frameshift mutation in ompK35 results in a truncated, 42-amino acids OmpK35, resulting in a nonfunctional porin . Transfer of wild-type ompK36 to KP1244 resulted in a 2-fold decrease in its MIC value for CAZ/AVI, from 32/4 to 16/4 mg/L, whereas transfer of wild-type ompK36 to KP1245 did not alter the MIC value. These findings indicate that although Arg191Leu does not affect CAZ/AVI MIC values, the Thr333Asn substitution alters susceptibility to CAZ/AVI. By contrast, transfer of wild-type ompK35 to KP1244 and KP1245 reduces their MIC values for CAZ/AVI by 8-fold, to 4/4 and 0.5 mg/L, respectively. A strain of Klebsiella oxytoca with a CAZ/AVI MIC value of 16/4 mg/L lacks the carbapenemase gene, whereas its OmpK36 has several amino acid substitutions (Ala192Pro and Asn229Ser) and deletions (Phe268 and 272_277GlyAspSerAspSerIle deletions; Aitken et al., 2016). Screening of two CAZ/AVI-resistant strains showed decreased ompK36 gene expression and a premature stop codon in ompK35, resulting in a truncated porin (Castanheira et al., 2017). An alteration in the L3 region of OmpK36 in K. pneumoniae results in decreased susceptibility to CAZ/AVI (Castanheira et al., 2020), indicating that impaired cell membrane permeability is associated with elevated MIC values to CAZ/AVI. Resistance of A. baumannii strains to CAZ/AVI is due mainly to the failure of AVI to penetrate the outer membrane (Mushtaq et al., 2010). In addition to OmpC, OmpF, and their homologous proteins, the introduction of a wild-type Lamb into resistant strains carrying a variant Lamb with Arg33His and Arg374Leu substitutions results in a 2-fold reduction in the MIC values of these strains for CAZ/AVI, indicating that variant Lamb contributes to resistance to CAZ/AVI .

| CAZ/AVI resistance mechanisms associated with efflux pumps
Overexpression of efflux pumps can reduce intracellular concentration of antibiotics and strain susceptibility to antibiotics (Nikaido & Pagès, 2012). Addition of the efflux pump inhibitor phenylalanine-arginine β-napthylamide (PaβN) and CAZ/AVI revealed that PaβN did not significantly affect the antibacterial activity of CAZ/AVI, showing that CAZ/AVI enters cells primarily through a passive barrier, with little contribution from active efflux AcrAB-TolC (Pagès et al., 2015). In contrast to Enterobacteriaceae, the presence of PAβN or the efflux pump inhibitor carbonyl cyanide mchlorophenylhydrazone (CCCP) reduces the MIC values of some strains of P. aeruginosa for CAV/AZI, from >32/4 to <0.06/4 mg/L (M. L. Winkler, Papp-Wallace, Hujer, et al., 2015).
Overexpression of the mexA, mexB, and oprM genes in CAZ/AVI-resistant strains of P. aeruginosa isolated from patients with cystic fibrosis altered their MIC values to CAV/AZI from 0.5/4 to 2/4 mg/L. Further analysis showed that the efflux rate of the efflux pump was associated with the MIC value. For example, the MIC values for CAV/AVI of isolates with very slow and faster efflux rates of the efflux pump were 8/4 and 64/4 mg/L, respectively. The mechanism underlying the resistance of these bacteria to CAV/AVI was is due mainly to increased activity of the MexAB-OprM efflux system, resulting from overexpression of mexA and overproduction of AmpC (Chalhoub et al., 2018). These findings suggest that efflux pump-related mutations play a non-negligible role in the mechanism of CAZ/AVI resistance.

| CAZ/AVI resistance mechanisms caused by variants of PBPs
The Leu169Pro amino acid substitution in PBP3 results in a significant increase in the MIC value for CAZ/AVI. Introduction of the wild-type ftsl gene into the resistant strain reduces the MIC value for CAZ/AVI from 128/4 to 4/4 mg/L . Alterations in the PBP-encoding gene ftsI are associated with CAZ/AVI resistance (Castanheira et al., 2019). A CAZ/AVI MIC of 8/4 μg/ml was reported against one E. coli isolate with an unusual TIPY insertion following Tyr344 in penicillin-binding protein 3 (PBP 3) as the result of gene duplication, which could affect the entry of CAZ (Zhang et al., 2017). In addition, some studies showed that a strain of K. pneumoniae was continuously cultured in broth medium with CAZ/AVI concentration of 1/4 mg/L under antibiotic selective pressure. The results showed that amino acid substitution of Asp354Ala of mdrA gene encoding PBP2 eventually increased MIC of CAZ/AVI from 0.25/4 to 8/4 mg/L (Karlowsky et al., 2021;Livermore et al., 2018).

| METHODS OF DETECTING CAZ/AVI RESISTANCE
Rapid detection of resistance to CAZ/AVI can be beneficial to the early implementation of appropriate therapeutic interventions. The rapid colorimetric method-Andrade screening antimicrobial test (ASAT), involving a 3 h incubation with Andrade solution, can rapidly detect bacterial resistance to CAZ/AVI, thereby enabling timely treatment. The ASAT reagent contains NaCl, peptone, and Andrade indicator, with a pH adjusted to 7.4 ± 0.2. Each reaction contains 175 μl of ASAT reagent and a certain concentration of CAZ/AVI, to which is added 25 μl of bacterial suspension, followed by incubation at 37 C for 3 h. Gram-negative bacteria are detected by fermentation of glucose medium. The presence of a purple-red color indicates a CAZ/AVI-resistant strain, whereas a light pink color indicates a CAZ/AVI-susceptible strain. A selective medium called Super-CAZ/AVI has also been developed to screen for CAZ/AVI-resistant Gram-negative bacteria. This assay was based on findings showing that the resistance breakpoints of Enterobacteriaceae and P. aeruginosa to CAZ/AVI are 8/4 mg/L, with the medium optimized to a final CAZ/AVI concentration of 6/4 mg/L. A diluted suspension of bacteria is layered onto culture medium containing CAZ/AVI, and bacterial growth is evaluated after 18 h. SuperCAZ/AVI medium can be used to screen patients for the presence of potentially CAZ/AVI-resistant strains, allowing the implementation of targeted measures to quickly limit the transmission of infection (Sadek et al., 2020). This culture medium can also be used in epidemiological investigations to assess the prevalence of CAZ/AVI-resistant Gram-negative bacteria in populations. The development of new methods of detecting CAZ/AVI resistance may facilitate more rapid detection and treatment of resistant strains. These methods to detect CAZ/AVI resistance are with excellent values of sensitivity and specificity. And these screening methods provide the opportunity to detect CAZ/AVI-resistant isolates regardless of their resistance mechanisms. They are reliable and economical. However, the specific resistance mechanisms to CAZ/AVI are unclear, so it cannot guide medication concretely.

| CONCLUSION
CAZ/AVI, a combination of β-lactam and a novel β-lactamase inhibitor (AVI), has been listed since 2015, which is an important drug used to treat complicated urinary tract infections and complicated intra-abdominal infections caused by carbapenem-resistant Gram-negative bacterial infections. Increases in its clinical use have led to the development of bacterial resistance. According to the global resistance surveillance reports in recent years, the overall resistance rate of Gram-negative bacteria to CAZ/AVI was low. The resistance rate of P. aeruginosa to CAZ/AVI was significantly higher than that of Enterobacteriaceae. In addition, the resistance rate of CAZ/AVI in carbapenem-resistant Gram-negative bacteria was significantly increased. And the resistance rate of CAZ/AVI was significantly different among different continents. By summarizing the mechanisms of resistance to CAZ/AVI, we find that β-lactamase variants, especially those associated with bla KPC gene mutations, are the most widely reported. Other possible mechanisms associated with CAZ/AVI resistance include overexpression of efflux pumps, reduced expression of outer membrane proteins, and gene mutations leading to loss of outer membrane proteins. Finally, we summarized the detection methods of CAZ/AVI resistance.
These studies on the resistance mechanisms to CAZ/AVI will help guide clinical drug use. Currently, Combination drug therapy is mostly used to prevent and treat the emergence of resistant strains to CAZ/AVI in clinical practice, but the treatment lacks specificity. At present, broad-spectrum metal β-lactamase inhibitors have been developed for the strains which are natural resistance to CAZ/AVI. As for the treatment of CAZ/AVI non-naturally resistant strains, research and development of specific medicine may be the trend of future development. Additionally, the development of new methods to detect CAZ/AVI resistance may be beneficial for the treatment and control of drug-resistant bacterial infections.

ACKNOWLEDGMENTS
We thank Chen Yunbo, Shen Ping, Ji Jinru, Ying Chaoqun, Liu Zhiying, and Chen Tao of the First Affiliated Hospital, College of Medicine, Zhejiang University for their helpful discussion.